The phagocyte NADPH oxidase generates superoxide which is essential for microbicidal activity, but which also has been implicated in inflammatory disease. The oxidase is comprised of at least five polypeptides: gp91-phox and p22-phox comprise the membrane associated cytochrome b558, and p47-phox and p67-phox and rac which are found in the cytosol of resting phagocytes. Activation of phagocytes triggers translocation of p47-phox, p67-phox and rac to the membrane and formation of a complex with cytochrome b558. The proposed studies will investigate how cell activation triggers conversion of the oxidase from a dormant to an active NADPH oxidase. At least three mechanisms regulate formation of NADPH oxidase 1) phosphorylation of p47-phox, 2) GDP/GTP cycling of rac, a member of the ras superfamily of GTPases, and 3) sequestration of soluble phox components in an inactive cytosol complex. It is proposed to 1) characterize the structure of the unphosphorylated inactive p47- phox and interactions of a p47-phox arginine/lysine-rich region with binding sites in p47-phox and other phox proteins. 2) Determine how phosphorylation at multiple p47-phox serines causes conversion to the active p47-phox conformation. 3) examine how rac activates p67-phox by dissociating p67-phox from a p40-phox/p67-phox complex and facilitates interaction of p67-phox with cytochrome b558. The overall approach is to identify interaction within and between phox proteins that are altered as a result of activation and assembly of the NADPH oxidase. Multiple methods will be used to characterize binding -two hybrid, affinity precipitation, fluorescence polarization, using recombinant phox proteins. The role of binding interactions will be examined in an in vitro assay of oxidase activity and in phox-deficient EBV-transformed B cells transfected with recombinant phox cDNA. A more detailed knowledge of the NADPH oxidase lead to new strategies for design of anti-inflammatory agents.